Breast Cancer is a leading cause of cancer death in the United States with ~200,000 new cases and ~40,000 deaths each year. A better understanding of the genetic effectors of tumor growth and suppression would enable the identification of new targets for therapy. The aims of this proposal are directed at identifying and validating key components of the TGF-beta signaling pathway that would lead to the development of specific therapies for the treatment of breast cancer. AIM 1: Analyzingthe role of cdc25A in TGF-beta mediated growth stimulation of breast epithelial cells. Our initial studies demonstrated that TGF- beta mediated growth stimulation correlated with an increased expression of the cdc25A tyrosine phosphatase gene in cells lacking p21. In this aim we will further analyze the ability of cdc25A to mediate TGF-beta induced cellular proliferation using RNA interference, promoter/mutational/ChIP assays, and p21 cDNA mutational studies, coupled with an analysis of human breast cancers using tissue microarrays. AIM 2: Identifying mediators of TGF-beta induced cellular proliferation. In Aim 2, we hypothesize that there are other integral genes whose upregulation is needed for TGF-beta induced growth stimulation. We will use microarray expression analysis to identify these genes using our previously characterized p21-/- cell lines. In order to validate that these genes are true mediators of TGF-beta stimulated cellular proliferation, RNA interference gene "knock down" experiments and targeted gene ablation via homologous recombination will be performed. AIM 3: Identifying genetic effectorsthat mediate the growth response to TGF-beta. In this aim, we will identify additional effectors of TGF-beta mediated growth inhibition using a functional genetic screen. We will combine exon trapping with genetic instability to isolate genes whose inactivation confers resistance to TGF-beta mediated growth suppression. Isolated genes will be verified and validated by restoration of the putative gene and correlated with immunohistochemical studies using tissue microarrays.